The present invention relates to the field of site directed therapy. Nowadays there are a number of methods of site directed therapy which have been suggested to eliminate unwanted cells or other biological matter, e.g., vascular occlusions, or infectious organisms from the body of a mammalian subject.
There are many fields of therapy in which such methods may be applied. The most important ones include, without limitation, immune diseases (either auto immune diseases or acquired immune diseases), cancer and viral or microbial infections.
Site directed therapy is a method whereby a cytotoxic compound is delivered to the immediate vicinity of the target cell or infectious organism. This is usually done by coupling a targeting moiety to the cytotoxic compound.
This targeting moiety recognizes a structure in, on, or near the target. Known targeting moieties include, but are not limited to, antibodies, more specifically monoclonal antibodies and more preferably human monoclonal antibodies, nucleic acids, receptor directed ligands and the like.
Cytotoxic compounds can be for instance drugs, such as adriamycin, toxins such as ricin A and radioisotopes.
Radioisotopes can be used not only for therapy, but also to identify the site or sites of the target (imaging). This invention provides methods of therapy and imaging using a conjugate of a targeting moiety and at least one radioisotope.
Therapy with targeting moieties is widely known. Targeting can be accomplished by aiming the targeting moiety directly to the wanted site, but it may also be directed to another targeting moiety which is directed to the wanted site (so called pretargeting). Pretargeting offers an advantage over direct targeting when the specificity of the targeting moieties is not sufficient. By using a first localizing moiety followed by a second one coupled to a cytotoxic compound, the amount of cytotoxic compound delivered to non-target sites can be lowered significantly.
Known targeting moieties, such as antibodies, often cannot be provided with a large amount of cytotoxic compounds without hampering their targeting specificity.
Therefore it has often been suggested to use a carrier molecule, such as HSA or a nucleic acid, or a polymer, which can be loaded with a high number of cytotoxic compounds and coupled to a targeting moiety.
All of the above-mentioned variations on the theme of site directed therapy and/or imaging can be used more advantageously with the present invention.
A well established problem in the field of imaging and site directed radiotherapy is to find a suitable radioisotope. Apart from the amount of energy that is released upon their decay, which should be sufficient to be measurable outside the subject in the case of imaging and sufficiently lethal to the target in the case of therapy, there is also a problem in finding an isotope with a suitable half-life.
An isotope with a long half life cannot be chosen because of the biological half life of the targeting moiety, which means that most of the isotopes will decay after disintegration of the conjugate. This decay after the disintegration of the conjugate will lead to cytotoxicity to other cells or tissues than the target.
Furthermore, all conjugates which do not localize will be secreted from the body and present a radio active waste problem.
It is also not practical to choose a radioisotope with too short a half life, because of packing and shipping delays and because the institution carrying out the therapy must be equipped to make the conjugate, transport it to the patient and administer it in a very short interval of time, otherwise most of the radioisotope will have decayed before entering the body, let alone localization at the target site.
The isotopes used for imaging usually are gamma emitting isotopes, for therapy auger electron emitting xcex1-xcex2-emitting, or xcex1-emitting isotopes may be used.
Most preferred for the present invention are xcex1-emitting isotopes.
The short-range cell-killing effect of xcex1-particles is enormous: a 1 mm diameter tumor, comprising maybe 600,000 cancer cells needs about 6 xcex1-particles of 6 MeV per cell to deliver a dose of 600 red, causing a 99.9% cell-kill ratio, and that specifically because of the stochastic nature of the hit- and kill-mechanism.
However, due to the same stochastic nature, a 10 times lower a-radiator dose will enhance the cell survival ratio with a factor 500: more than 50% of the cells (or non-tumor cells in similar morphology for that matter) would survive a 60 red xcex1-radiation dose, equivalent to 0.6 xcex1-particles per cell.
This characteristic would make an effective xcex1-radioimmunotherapy within reach, provided that a xe2x80x9cquality factorxe2x80x9d for the isotope-antibody conjugate of 10 or better can be obtained. It is the purpose of the present invention to contribute towards this goal in a most essential manner.
The quality factor is a ratio between localized antibody at the target site, divided by the antibody xe2x80x9cstickingxe2x80x9d to other tissue.
The notion of using xcex1-particle emitting radioisotopes as agents for the killing of tumor cells was already mentioned in the literature during the mid-fifties. Since then other potential candidate isotopes were and are being proposed, of which a good summary is given by Fisher (1) and by Wilbur (2) which brings the list to (with their half-lives between parentheses): 223Ra (11.4 d), 225Ac (10 d), 224Ra (3.6 d), 225Fm (20 h) 211At (7,2 h), 212Bi (60 m), and 213Bi (47 m).
Although important publications appear regularly in the literature regarding microdosimetry, antibody isotope coupling techniques, pre-clinical in vitro and in vivo experiments, no clearly defined, larger scale clinical experiments are being done until now, for a variety of reasons:
a. very few human monoclonal antibodies with proven sufficient quality are available yet,
b. very limited biological safety data are available for antibody coupling agent (the latter for the binding of the radioisotope) combinations,
c. some isotopes may not become available for large scale application at acceptable cost prices (225Fm),
d. isotopes may be too difficult and therefore too expensive to obtain because of the necessary procurement process (211At from 209Bi by a (xcex1, 2n) reaction in a cyclotron and subsequent isolation plus purification),
e. other isotopes do have a Rn-isotope as first daughter in their decay sequence, allowing redistribution of daughter nuclei before decay (224Ra, 223Ra), and also necessitating gas-tight reaction conditions,
f. some isotopes may have a relatively long living daughter isotope somewhere in their decay sequence (224Ra, 223Ra, 225Ac) also with the chance that daughters thereof may redistribute before decay,
g. the radioactive halflife of some isotopes is so long that most of the activity leaves the patient undecayed, resulting in a waste problem (223Ra, 224Ra 225AC) or
h. the halflife of the isotope is so short that most of the isotope decays before reaching its ultimate target (212Bi, 213Bi),
i. sufficient precursor material may not be available to extract at one time the necessary amount of isotope for a single patient treatment (212Bi, 213Bi), and,
j. isotopes producing hard gamma rays in their isotope decay need shielding facilities to prevent radiation hazards to technicians and nursing personnel.
One or more of the arguments listed above will make it very difficult, if not impossible for some of the isotopes to ever be used on a large scale for xcex1-radio-immunotherapy, and that in particular if one or more of the others can be used on acceptable technical, logistical and financial conditions.
The closest prior art to the present invention (the French patent application FR-A-2 527 928) discloses a method to produce conjugates of an antibody and 212Bi. However, these conjugates still suffer from the drawbacks mentioned above under e, h, i and j.
The present invention provides a therapeutic radioconjugate of a targeting moiety bound, directly or indirectly, to at least one relatively short lived xcex1-particle emitting radioisotope, the targeting moiety being effective to deliver the conjugate to a pre-determined pathological site in a mammal. The pathological site comprises a target moiety, which together with the targeting moiety, constitutes a specific binding pair.
The targeting moiety is preferably selected from the group consisting of an antibody, or antibody fragment which bind specifically to a cell-surface antigen; or a ligand or ligand fragment which bind specifically to a cell surface receptor.
The pathological site may comprise diseased cells, e.g., tumor cells, cells at an inappropriate site, e.g., neutrophils at site of inflammation, platelets at sites of injury, or it may be an extracellular structure, such as fibrin or thrombin within a coronary vessel at a site of infarct, cholesterol at a site of atherosclerotic placque, calcification in diseased vessels, amyloid plaque at disease sites in the brain, or the like.
Also according to this invention, a method is provided for preparing the above-identified therapeutic radioconjugate. The method involves loading onto a binding medium a relatively long lived radioisotope, the decay sequence of said relatively long lived radioisotope yielding daughter isotopes that emit predominantly xcex1- and/or xcex2-particles without harmful gamma radiation, eluting said xcex1-particle emitting radioisotope from said binding medium and coupling said xcex1-particle emitting radioisotope, substantially free of said relatively long lived radioisotope, to said targeting moiety.
Relatively long lived means that the radioisotopes have a decay time in the order of several days, which enables sufficient time for packing and shipping. Relatively short lived in this context means that the radioisotopes have a decay time in the order of minutes or hours.
The reference to a decay sequence in which predominantly xcex1 and/or xcex2-rays are emitted is meant to signify a decay sequence which does not cause danger for radiation hazards caused by gamma rays to people working with the compounds without the burden of applying protective shielding.
Preferably, the relatively long lived isotope is 225Ac and the relatively short lived xcex1-particle emitting radioisotope is 213Bi.